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Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O2 to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic analysis revealed that the k cat, K m, and k cat/K m of nLcc4 with substrate ABTS were 3,382 s -1, 65.0 ± 6.5 μM, and 52 s -1μM-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s -1, 56.7 ± 3.2 μM, and 0.004 s -1μM-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.

The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1) encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+)-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+)-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

V. cholerae can form sessile biofilms associated with abiotic surfaces, cyanobacteria, zoo-plankton, mollusks, or crustaceans. Along with the vibrio polysaccharide, secreted proteins of the rbm gene cluster are key to the biofilm ultrastructure. Here we provide a thorough structural characterization of RbmA, a protein involved in mediating cell-cell and cell-biofilm contacts. We correlate our structural findings with initial ligand specificity screening results, NMR protein-ligand interaction analysis, and complement our results with a full biocomputational study.